Rapid preparation of nuclei-depleted detergent-resistant membrane fractions suitable for proteomics analysis

<p>Abstract</p> <p>Background</p> <p>Cholesterol-rich membrane microdomains known as lipid rafts have been implicated in diverse physiologic processes including lipid transport and signal transduction. Lipid rafts were originally defined as detergent-resistant membranes (DRMs) due to their relative insolubility in cold non-ionic detergents. Recent findings suggest that, although DRMs are not equivalent to lipid rafts, the presence of a given protein within DRMs strongly suggests its potential for raft association in vivo. Therefore, isolation of DRMs represents a useful starting point for biochemical analysis of lipid rafts. The physicochemical properties of DRMs present unique challenges to analysis of their protein composition. Existing methods of isolating DRM-enriched fractions involve flotation of cell extracts in a sucrose density gradient, which, although successful, can be labor intensive, time consuming and results in dilute sucrose-containing fractions with limited utility for direct proteomic analysis. In addition, several studies describing the proteomic characterization of DRMs using this and other approaches have reported the presence of nuclear proteins in such fractions. It is unclear whether these results reflect trafficking of nuclear proteins to DRMs or whether they arise from nuclear contamination during isolation. To address these issues, we have modified a published differential detergent extraction method to enable rapid DRM isolation that minimizes nuclear contamination and yields fractions compatible with mass spectrometry.</p> <p>Results</p> <p>DRM-enriched fractions isolated using the conventional or modified extraction methods displayed comparable profiles of known DRM-associated proteins, including flotillins, GPI-anchored proteins and heterotrimeric G-protein subunits. Thus, the modified procedure yielded fractions consistent with those isolated by existing methods. However, we observed a marked reduction in the percentage of nuclear proteins identified in DRM fractions isolated with the modified method (15%) compared to DRMs isolated by conventional means (36%). Furthermore, of the 21 nuclear proteins identified exclusively in modified DRM fractions, 16 have been reported to exist in other subcellular sites, with evidence to suggest shuttling of these species between the nucleus and other organelles.</p> <p>Conclusion</p> <p>We describe a modified DRM isolation procedure that generates DRMs that are largely free of nuclear contamination and that is compatible with downstream proteomic analyses with minimal additional processing. Our findings also imply that identification of nuclear proteins in DRMs is likely to reflect legitimate movement of proteins between compartments, and is not a result of contamination during extraction.</p

The IDENTIFY study: the investigation and detection of urological neoplasia in patients referred with suspected urinary tract cancer - a multicentre observational study

Objective To evaluate the contemporary prevalence of urinary tract cancer (bladder cancer, upper tract urothelial cancer [UTUC] and renal cancer) in patients referred to secondary care with haematuria, adjusted for established patient risk markers and geographical variation. Patients and Methods This was an international multicentre prospective observational study. We included patients aged ≥16 years, referred to secondary care with suspected urinary tract cancer. Patients with a known or previous urological malignancy were excluded. We estimated the prevalence of bladder cancer, UTUC, renal cancer and prostate cancer; stratified by age, type of haematuria, sex, and smoking. We used a multivariable mixed-effects logistic regression to adjust cancer prevalence for age, type of haematuria, sex, smoking, hospitals, and countries. Results Of the 11 059 patients assessed for eligibility, 10 896 were included from 110 hospitals across 26 countries. The overall adjusted cancer prevalence (n = 2257) was 28.2% (95% confidence interval [CI] 22.3–34.1), bladder cancer (n = 1951) 24.7% (95% CI 19.1–30.2), UTUC (n = 128) 1.14% (95% CI 0.77–1.52), renal cancer (n = 107) 1.05% (95% CI 0.80–1.29), and prostate cancer (n = 124) 1.75% (95% CI 1.32–2.18). The odds ratios for patient risk markers in the model for all cancers were: age 1.04 (95% CI 1.03–1.05; P < 0.001), visible haematuria 3.47 (95% CI 2.90–4.15; P < 0.001), male sex 1.30 (95% CI 1.14–1.50; P < 0.001), and smoking 2.70 (95% CI 2.30–3.18; P < 0.001). Conclusions A better understanding of cancer prevalence across an international population is required to inform clinical guidelines. We are the first to report urinary tract cancer prevalence across an international population in patients referred to secondary care, adjusted for patient risk markers and geographical variation. Bladder cancer was the most prevalent disease. Visible haematuria was the strongest predictor for urinary tract cancer

Operationalising the Family-Friendly Medical Workplace and the Development of FAM-MED, a Family-Friendly Self-Audit Tool for Medical Systems: A Delphi Consensus

Globally, the call for Family-Friendly (FF) workplaces is loud and clear. However, this call is inaudible in medical workplaces, despite both well-established benefits of FF workplaces across businesses and well-known effects of work–family conflict on the well-being and practice of doctors. We aimed to use the Delphi consensus methodology to: (i) operationalise the Family-Friendly medical workplace and (ii) develop a Family-Friendly Self-Audit tool for medical workplaces. The expert medical Delphi panel was deliberatively recruited to capture a breadth of professional, personal, and academic expertise, diversity of age (35–81), life stage, family contexts and lived experience of dual commitments to work and family, and diversity of work settings and positions. Results reflected the inclusive and dynamic nature of the doctor’s family and the need to adopt a family life cycle approach to FF medical workplaces. Key processes for implementation include holding firms to zero discrimination; flexibility and openness to dialogue and feedback; and a mutual commitment between the doctor and the department lead to best meet the doctor’s individualised needs while still ensuring optimal patient care and team support and cohesion. We hypothesise that the Department Head may be the key to implementation but recognise the workforce constraints to realising these aspirational systemic shifts. It is time we acknowledge that doctors have families, to narrow the gap between identifying as a partner, mother, father, daughter, son, grandparent, and identifying as a doctor. We affirm the right to be both good doctors and good family members

Operationalising the Family-Friendly Medical Workplace and the Development of FAM-MED, a Family-Friendly Self-Audit Tool for Medical Systems: A Delphi Consensus

From MDPI via Jisc Publications RouterHistory: received 2023-05-17, collection 2023-06, rev-recd 2023-06-02, accepted 2023-06-04, epub 2023-06-07Peer reviewed: TrueArticle version: VoRPublication status: PublishedGlobally, the call for Family-Friendly (FF) workplaces is loud and clear. However, this call is inaudible in medical workplaces, despite both well-established benefits of FF workplaces across businesses and well-known effects of work–family conflict on the well-being and practice of doctors. We aimed to use the Delphi consensus methodology to: (i) operationalise the Family-Friendly medical workplace and (ii) develop a Family-Friendly Self-Audit tool for medical workplaces. The expert medical Delphi panel was deliberatively recruited to capture a breadth of professional, personal, and academic expertise, diversity of age (35–81), life stage, family contexts and lived experience of dual commitments to work and family, and diversity of work settings and positions. Results reflected the inclusive and dynamic nature of the doctor’s family and the need to adopt a family life cycle approach to FF medical workplaces. Key processes for implementation include holding firms to zero discrimination; flexibility and openness to dialogue and feedback; and a mutual commitment between the doctor and the department lead to best meet the doctor’s individualised needs while still ensuring optimal patient care and team support and cohesion. We hypothesise that the Department Head may be the key to implementation but recognise the workforce constraints to realising these aspirational systemic shifts. It is time we acknowledge that doctors have families, to narrow the gap between identifying as a partner, mother, father, daughter, son, grandparent, and identifying as a doctor. We affirm the right to be both good doctors and good family members

Operationalising the Family-Friendly Medical Workplace and the Development of FAM-MED, a Family-Friendly Self-Audit Tool for Medical Systems: A Delphi Consensus.

From Europe PMC via Jisc Publications RouterHistory: ppub 2023-06-01, epub 2023-06-07Publication status: PublishedGlobally, the call for Family-Friendly (FF) workplaces is loud and clear. However, this call is inaudible in medical workplaces, despite both well-established benefits of FF workplaces across businesses and well-known effects of work-family conflict on the well-being and practice of doctors. We aimed to use the Delphi consensus methodology to: (i) operationalise the Family-Friendly medical workplace and (ii) develop a Family-Friendly Self-Audit tool for medical workplaces. The expert medical Delphi panel was deliberatively recruited to capture a breadth of professional, personal, and academic expertise, diversity of age (35-81), life stage, family contexts and lived experience of dual commitments to work and family, and diversity of work settings and positions. Results reflected the inclusive and dynamic nature of the doctor's family and the need to adopt a family life cycle approach to FF medical workplaces. Key processes for implementation include holding firms to zero discrimination; flexibility and openness to dialogue and feedback; and a mutual commitment between the doctor and the department lead to best meet the doctor's individualised needs while still ensuring optimal patient care and team support and cohesion. We hypothesise that the Department Head may be the key to implementation but recognise the workforce constraints to realising these aspirational systemic shifts. It is time we acknowledge that doctors have families, to narrow the gap between identifying as a partner, mother, father, daughter, son, grandparent, and identifying as a doctor. We affirm the right to be both good doctors and good family members

Dynamic reciprocity in cell–scaffold interactions

Tissue engineering in urology has shown considerable promise. However, there is still much to understand, particularly regarding the interactions between scaffolds and their host environment, how these interactions regulate regeneration and how they may be enhanced for optimal tissue repair. In this review, we discuss the concept of dynamic reciprocity as applied to tissue engineering, i.e. how bi-directional signaling between implanted scaffolds and host tissues such as the bladder drives the process of constructive remodeling to ensure successful graft integration and tissue repair. The impact of scaffold content and configuration, the contribution of endogenous and exogenous bioactive factors, the influence of the host immune response and the functional interaction with mechanical stimulation are all considered. In addition, the temporal relationships of host tissue ingrowth, bioactive factor mobilization, scaffold degradation and immune cell infiltration, as well as the reciprocal signaling between discrete cell types and scaffolds are discussed. Improved understanding of these aspects of tissue repair will identify opportunities for optimization of repair that could be exploited to enhance regenerative medicine strategies for urology in future studies